Linear tandem antigen epitope polypeptide of African swine fever virus RAN polymerase and application of linear tandem antigen epitope polypeptide
By preparing linear tandem antigenic epitope peptides of ASFV RAN polymerase, the problem of insufficient protective efficacy of ASF vaccines under highly variable conditions was solved, achieving effective T-cell immune response and viral clearance, and providing a basis for peptide vaccines and diagnostic agents.
Patent Information
- Application Number
- CN202511677297.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-17
- Publication Date
- 2026-02-10
AI Technical Summary
Existing ASF vaccines have insufficient protective efficacy due to their high variability, especially after type II strains are converted into type I/II recombinant strains, which lack effective cross-protection capabilities. Furthermore, the role of T-cell immune responses in host resistance to ASFV infection is not fully utilized.
Linear tandem antigenic epitope peptides of ASFV RAN polymerase were prepared. Recombinant vectors were designed and expressed and purified using linear tandem antigenic epitope peptides NP1249L-1, H359L-1, and H359L-2. Antigen-presenting cells were then prepared for the preparation of peptide vaccines and diagnostic reagents.
It effectively stimulates specific T-cell immune responses, assists in controlling ASFV infection and viral clearance, and provides a theoretical basis for ASFV peptide vaccines and diagnostic agents.
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Figure CN121495899A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of biotechnology, and particularly relates to a linear tandem antigen epitope polypeptide of African swine fever virus RAN polymerase and application. BACKGROUND
[0002] African swine fever (ASF) is an acute, fever, and highly contagious disease caused by African swine fever virus (ASFV) infection in susceptible hosts such as domestic pigs and European wild boars. The infected pigs show symptoms such as high fever, hemorrhage, anorexia, and dyspnea, and the mortality rate can be as high as 100%. Due to the lack of effective treatment methods, clinical veterinarians mainly adopt measures such as isolation, culling, and harmless treatment to control the epidemic. According to the ASF epidemic investigation report, ASF epidemic prevention and control still faces serious pressure, and the epidemic is still in a state of continuous spread.
[0003] ASFV is a double-stranded DNA virus with a genome size of about 190 kb, containing 151-167 open reading frames, encoding more than 150 proteins, of which about 50 are structure-related proteins, and also encoding RAN polymerase, DAN replicase, topoisomerase, and other key enzymes supporting the life cycle of ASFV replication, and also encoding functional factors containing various regulatory host immune responses. However, the functions of about half of the ASFV gene-encoded proteins are still unclear. It was previously believed that the immune protection effect induced by the vaccine mainly depends on the neutralizing antibodies triggered by the outer or easily exposed proteins, therefore, the development of ASF vaccine mainly focuses on structural proteins such as P72 protein, P54 protein, P30 protein, etc., which play a protective role by inducing neutralizing antibodies to block virus transmission. However, in recent years, the protection of subunit vaccines developed based on the above-mentioned proteins in the clinic needs to be further improved, especially under the high variability of ASFV, the epidemic strain in China's pig population has also changed from type II strain to I / II recombinant strain, which makes the development of ASF vaccine products also need to consider the cross-protection ability. On the basis of the above research, researchers found that the OURT88 / 3 and NH / P68 modified strain vaccines provide 100% protection against homologous or heterologous strain challenge after immunization, and the protection disappears after interfering with CD8 + T cell function, indicating that T cell immune response plays a key role in host resistance to ASFV infection.
[0004] ASFV encodes its own eukaryotic-like RNA polymerase, the core structure of which shares conservation with the 12 subunits of eukaryotic RNA polymerase II (Pol II) and the RNA polymerases of other nucleocytoplasmic large DNA viruses (NCLDVs). As an indispensable component of the viral replication and gene expression machinery, ASFV RNA polymerase precisely regulates the expression of viral genes, affecting the replication efficiency, pathogenicity, immune evasion strategies, and remodeling of host cell metabolism within the host cell by encoding its own transcription machinery. Therefore, focusing on the ASFV RNA polymerase T cell epitope to develop vaccines and other products that induce specific T cell responses not only helps to reveal the complex life cycle of ASFV, which is of great significance for the development of safe and effective ASF polypeptide vaccines based on T cell antigen epitopes, but also provides a key target for the development of effective antiviral strategies and drugs. SUMMARY
[0005] The problem to be solved by the present application is to prepare an ASFV RAN polymerase protein recombinant T cell epitope antigen to effectively stimulate specific T cell immune response and exert antiviral efficacy.
[0006] To solve the above problems, the technical scheme of the present application is as follows: The primary object of the present application is to provide an African swine fever virus RAN polymerase linear tandem antigen epitope polypeptide, wherein the amino acid sequence of the linear tandem antigen epitope polypeptide is shown in SEQ ID NO. 4 or SEQ ID NO. 5.
[0007] Preferably, the linear tandem antigen epitope polypeptide is obtained by linearly connecting one or more epitope polypeptides NP1249L-1, epitope polypeptide H359L-1 and epitope polypeptide H359L-2.
[0008] Preferably, the amino acid sequence of the epitope polypeptide NP1249L-1 is shown in SEQ ID NO. 1, the amino acid sequence of the epitope polypeptide H359L-1 is shown in SEQ ID NO. 2, and the amino acid sequence of the epitope polypeptide H359L-2 is shown in SEQ ID NO. 3.
[0009] The second object of the present application is to provide a nucleic acid molecule capable of encoding the linear tandem antigen epitope polypeptide.
[0010] The third object of the present application is to provide a recombinant vector containing the nucleic acid molecule.
[0011] The fourth object of the present application is to provide an antigen-presenting cell, which is sensitized by the linear tandem antigen epitope polypeptide.
[0012] The fifth object of the present application is to provide an African swine fever virus antigen prepared by coupling the linear tandem antigen epitope polypeptide with a carrier protein.
[0013] The sixth object of the present application is to provide the use of the linear tandem antigen epitope polypeptide for preparing a polypeptide vaccine against African swine fever virus, preparing a specific antibody against African swine fever virus, preparing a reagent or kit for diagnosing or detecting African swine fever virus.
[0014] The present application provides a linear tandem antigen epitope polypeptide of African swine fever virus RAN polymerase, the amino acid sequence of the linear tandem antigen epitope polypeptide is shown in SEQ ID NO. 4 or SEQ ID NO. 5, the linear tandem antigen epitope polypeptide is linearly connected by epitope polypeptide NP1249L-1, epitope polypeptide H359L-1 and epitope polypeptide H359L-2, the epitope polypeptide NP1249L-1, the epitope polypeptide H359L-1 and the epitope polypeptide H359L-2 are screened, have the functions of inducing ASFV specific T cells, assisting in controlling ASFV infection and virus clearance, and the experimental results show that the epitope polypeptide has the ability to induce ASFV specific T cell response, and provides a theoretical basis for subsequent development of polypeptide vaccines and diagnostic preparations based on ASFV protein derived epitopes. BRIEF DESCRIPTION OF DRAWINGS
[0015] Figure 1 It is an SDS-PAGE electrophoretogram of the ASFV RAN polymerase protein recombinant T cell epitope antigen purified in the present application.
[0016] Figure 2 It is a spot map of specific T cells and a statistical map thereof detected by using an ELISPOT experiment targeting IFN-γ for inducing specific T cells in vitro by the P6 antigen in the present application.
[0017] Figure 3 It is a spot map of specific T cells and a statistical map thereof detected by using an ELISPOT experiment targeting IFN-γ for inducing specific T cells in vitro by the P8 antigen in the present application.
[0018] Figure 4 It is a statistical map of cell activity detected by using CCK-8 for stimulating specific T cell proliferation by the P6 antigen in the present application.
[0019] Figure 5 It is a statistical map of cell activity detected by using CCK-8 for stimulating specific T cell proliferation by the P8 antigen in the present application. DETAILED DESCRIPTION
[0020] To make the objectives, technical solutions and advantages of the present invention clearer, the following will provide further description in conjunction with the embodiments. Unless otherwise specified, the experimental reagents, experimental equipment and experimental materials involved in the present invention are all commonly used or commercially available products in the field, and the terms and abbreviations involved have the conventional meanings in the field, such as IFN-γ (gamma interferon).
[0021] Experimental methods not specifically described in the following examples are generally performed under standard conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the product manufacturer.
[0022] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be used in this invention. The preferred embodiments and materials described herein are for illustrative purposes only.
[0023] The term "pharmaceutically acceptable carrier" refers to a carrier used for the administration of a therapeutic agent, meaning a pharmaceutical carrier that does not induce antibodies harmful to the individual receiving the composition and does not cause excessive toxicity after administration. These carriers are well known to those skilled in the art. Such carriers include, but are not limited to, saline, buffer solutions, glucose, water, glycerol, ethanol, adjuvants, and combinations thereof.
[0024] Pharmaceutically acceptable carriers in pharmaceutical compositions may contain liquids such as water, saline, glycerol, and ethanol. Additionally, these carriers may contain auxiliary substances such as wetting agents or emulsifiers, pH buffers, etc.
[0025] Typically, therapeutic compositions can be formulated as injectable preparations, such as liquid solutions or suspensions; they can also be formulated as solid forms suitable for reconstitution into solutions or suspensions or as liquid carriers prior to injection. Once formulated into the pharmaceutical compositions of the present invention, they can be administered directly to the subject. Therapeutic or prophylactic pharmaceutical compositions (including vaccines) containing the recombinant T-cell epitope antigen of the ASFV RAN polymerase protein of the present invention can be administered orally, subcutaneously, intradermally, or intravenously, and the therapeutic dosage regimen can be a single-dose regimen or a multiple-dose regimen.
[0026] Example 1: Construction of recombinant T-cell epitopes of ASFV RNA polymerase protein 1. Design of recombinant T-cell epitopes for ASFV RNA polymerase protein This invention provides epitope polypeptides of any one of the following ASFV RAN polymerase protein recombinant T cell epitope antigens: NP1249L-1 (number 7030), H359L-1 (number 7031), and H359L-2 (number 7032), whose amino acid sequences are SEQ ID No. 1, SEQ ID No. 2, and SEQ ID No. 3, respectively. Based on this, the three polypeptides can be used to design recombinant epitope antigens in any combination. Here, the three antigens are designed by repeating and tandemly using three T epitope antigens, or by repeating three times as a unit of tandemly using three T epitope antigens. Common protein linkers (such as GSGGSG) are used to connect the T cell epitopes, the T cell epitopes and the tag, and the T epitope antigens and the targeting peptide, respectively, and are designated as P6 and P8, with sequences shown in SEQ ID No. 4 and SEQ ID No. 5.
[0027] SEQ ID No. 1: ASGIPIIIRIY SEQ ID No. 2: EEQLPLVPVNV SEQ ID No. 3: NPFIKNAAKKLFQL SEQ ID No.4: HHHHHHEEQLPLVPVNVGGGSNPFIKNAAKKLFQLGGGSASGIPIIIRIYGGGSEEQLPLVPVNVGGGSNPFIKNAAKKLFQLGGGSASGIPIIIRIYGGGSEEQLPLVPVNVGGGSNPFIKNAAKKLFQLGGGSASGI PIIIRIYGGGSMRLLLLTFLGVCCLTPWVVEGVGTEVLEESSCVNLQTQRLPVQKIKTYIIWEGAMRAVIFVTKRGLKICADPEAKWVKAAIKTVDGRASTRKNMAETVPTGAQRSTSTAITLTGGSGGSGAHIVMVDAYKPTK SEQ ID No.5: HHHHHHEEQLPLVNVGGGSEEQLPLVPVNVGGGSEEQLPLVPVNVGGGSNPFIKNAAKKLFQLGGGSNPFIKNAAKKLFQLGGGSNPFIKNAAKKLFQLGGGSASGIPIIIRIYGGGSASGIPIIIRIYGGGSASGI PIIIRIYGGGSMRLLLLTFLGVCCLTPWVVEGVGTEVLEESSCVNLQTQRLPVQKIKTYIIWEGAMRAVIFVTKRGLKICADPEAKWVKAAIKTVDGRASTRKNMAETVPTGAQRSTSTAITLTGGSGGSGAHIVMVDAYKPTK 2. Expression and purification of recombinant T-cell epitope peptides from ASFV RNA polymerase protein The epitope peptides NP1249L-1 (number 7030), H359L-1 (number 7031), and H359L-2 (number 7032) mentioned in this invention were all synthesized by Wuhan Dangang Biotechnology Co., Ltd. All peptides had a purity higher than 95% and were correctly identified by mass spectrometry. All peptides were dissolved and stored at a final concentration of 1 mg / mL at -80°C for future use.
[0028] Based on the specific amino acid sequence of the obtained recombinant T cell epitope peptide, Suzhou Genewise Biotechnology Co., Ltd. synthesized and inserted the obtained recombinant gene sequence between BamHI and HindIII in the pET-28a vector to obtain recombinant expression vectors, which were named pET-28a-p6 and pET-28a-p8, respectively.
[0029] The above-mentioned pET-28a-p6 and pET-28a-p8 recombinant expression vectors were transformed into Escherichia coli BL21(DE3) competent cells to obtain the corresponding expression strains. The P6 and P8 fusion proteins were expressed using the Escherichia coli expression system. The expression method was as follows: a single colony was picked and inoculated into 5 ml of LB liquid medium (containing 100 μg / mL kanamycin) and cultured in a shaker at 37°C and 220 rpm for 12-18 h. The next day, the bacterial culture was inoculated into 1L of LB medium (containing 100μg / ml kanamycin) at a 1:100 ratio for expansion. When the OD600 reached 0.6–0.8, isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.5mmol / L. Expression was induced at 16℃ and 220r / min for 16–20h. The cultured bacterial culture was centrifuged at 4200rpm and 4℃ for 30min. The bacterial cells were resuspended in 0.01mol / L PBS buffer at 1g / ml, homogenized, and the supernatant was collected by centrifugation. The protein was purified by Ni column purification, and the imidazole in the protein sample was removed by dialysis to obtain two recombinant T epitope antigens, which were stored at -20℃ for later use. 20μL of samples before and after induction and at each purification stage were subjected to SDS-PAGE electrophoresis after heat denaturation for verification.
[0030] 3. Results Electrophoresis results as follows Figure 1 As shown, P6 and P8 were successfully induced to express in the bacterial cells, with a gel image size of approximately 30 kDa, consistent with the expected size. After affinity purification using a nickel column, high-purity P6 and P8 could be obtained by washing with 300 mM imidazole.
[0031] Example 2: ASFV RNA polymerase protein recombinant T cell epitope antigen stimulation of T cell activation 1. Separation of PBMCs The P6 and P8 antigens were coupled to the surface of ferritin nanoparticles (using the previously reported SpyTag / SpyCatcher technology), and then emulsified with an appropriate amount of adjuvant to prepare the vaccine. Three healthy 60-70 day old pigs that tested negative for ASFV were vaccinated with both vaccines via intramuscular injection in two separate doses, with a booster 14 days after the first immunization. Peripheral blood anticoagulated at 14 days post-second immunization was collected, and PBMCs were isolated within 4 hours according to the instructions of the porcine peripheral blood mononuclear cell isolation kit from Tianjin Haoyang Biological Products Technology Co., Ltd. Red blood cells were removed from the collected PBMCs using erythrocyte lysis buffer (TIANGEN). The cells were resuspended in RPMI 1640 complete medium (containing 10% FBS and a 1% penicillin-streptomycin-amphotericidal mixture), and 20 μL of the medium was mixed with an equal volume of trypan blue staining solution to label the cells. The viable cell count was then determined using a cell counter.
[0032] The vaccine preparation and immunization of the control group epitope peptides NP1249L-1 (number 7030), H359L-1 (number 7031), and H359L-2 (number 7032) were the same as described above.
[0033] 2. ELISpot assay to evaluate the ability of T cell epitope peptides to induce IFN-γ release. Pre-coat 96-well plates according to the Porcine IFN-γ (ALP) kit (Mabtech, 3130-2A) instructions. Place the prepared PBMCs (3-5 × 10⁶ per well) into each well. 5 Cells were added to 96-well plates coated with IFN-γ capture antibody, followed by the addition of T epitope peptides (final concentration 10 μg / mL) and controls (complete culture medium), and incubated in a cell culture incubator for 17-24 h. Subsequent ELISpot assays were performed according to the product instructions, and the final results were obtained using an ELISpot analyzer (Mabtech ASTOR 2).
[0034] 3. Experimental Results The results are as follows Figure 2 , Figure 3 As shown, compared to the control group, treatment with 10 μg / mL epitope peptides and antigens both demonstrated the ability to induce an increase in IFN-γ. Among them, recombinant T epitopes P6 and P8 showed the strongest stimulatory ability, with effects exceeding those of single epitope treatment. Treatment with 10 μg / mL peptides and antigens both stimulated IFN-γ release, with P6 showing the best effect, followed by P8. However, the induction effect of the P6 and P8 treatment groups was approximately 5 times higher than that of the single epitope peptide treatment group. Therefore, the T-cell epitope peptide antigen of the ASFV RNA polymerase protein and / or subunits mentioned in this invention has been verified through in vitro experiments to possess the ability to induce T cells to secrete IFN-γ, thus assisting in the control of ASFV infection and viral clearance.
[0035] Example 3: ASFV RNA polymerase protein recombinant T cell epitope peptide induces specific T cell proliferation 1. Isolation and preparation of porcine BMDCs and spleen lymphocytes Take the femur and tibia from the hind leg of a healthy 60-70 day old pig that is negative for ASFV. Remove the surface tissue from the bones, soak them in 75% absolute ethanol for 5 min, and then rinse three times with sterile PBS. Cut the bones and wash the bone marrow tissue with RPMI 1640 complete medium. Filter the bone marrow wash through a sterile 70 μm filter and collect the wash fluid. Centrifuge at 1500 rpm for 5 min and discard the supernatant. Add 3-5 mL of erythrocyte lysis buffer to remove erythrocytes, centrifuge at 1500 rpm for 5 min to collect the cells, resuspend the cells in 5 mL of RPMI 1640 complete medium, mix 20 μL with an equal volume of trypan blue staining solution to label the cells, and count the viable cells using a cell counter. Plant the cells at a density of 2 × 10⁶ cells per 100 mm cell culture dish. 7 The cells are evenly distributed on the plate.
[0036] BMDCs were induced using RPMI1640 complete medium containing 20 ng / mL porcine GM-CSF and 10 ng / mL porcine IL-4. After 3 days of culture, the medium was replaced with the same medium. On day 5, the differentiation level of BMDCs was observed under a microscope. After induction was completed, the medium was replaced with RPMI1640 complete medium.
[0037] Spleen tissue was taken from the same pig from which BMDCs were isolated. The spleen tissue was washed with sterile PBS under sterile conditions. Three 2cm × 2cm × 2cm pieces of spleen tissue were randomly cut, and the tissue was ground after adding porcine spleen lymphocyte separation medium from Tianjin Haoyang Biological Products Technology Co., Ltd. The mixture was then filtered through a sterile 70μm filter and collected at 800× [a certain temperature range]. g Centrifuge for 25 min, transfer the lymphocyte layer to a new 15 mL centrifuge tube, add 3-5 mL of erythrocyte lysis buffer to remove erythrocytes, centrifuge at 1500 rpm for 5 min to collect cells, resuspend the cells in 5 mL of RPMI 1640 complete culture medium containing 10 ng / mL porcine IL-2, mix 20 μL of the medium with an equal volume of trypan blue staining solution to label the cells, and count the number of viable cells using a cell counter. Culture the cells at a rate of 5 × 10⁶ cells per T25 cell culture flask. 6 After the cells were evenly distributed, they were placed in a cell culture incubator for later use.
[0038] 2. Preparation of ASFV RNA polymerase protein recombinant T cell epitope peptide-specific T cells The BMDCs prepared above were used in a 6-well plate with 6 × 10⁶ cells per well. 5 After seeding cells, the cells were allowed to rest overnight in a cell culture incubator. After the first replacement with RPMI 1640 medium at a final concentration of 10 μg / mL, the medium was replaced with the same medium every two days. After 3-5 stimulations, antigen-pulse-stimulated BMDCs were obtained.
[0039] Take the above 1×10 7One isolated spleen lymphocyte was placed in a 1 mL centrifuge tube, and 10–25 μL of Pig CD8a-PE (clone: 76-2-11) antibody was added. After incubation at 4°C for 30 min, IgG magnetic beads were added according to the recommended protocol in the CST product instructions, and the mixture was incubated at room temperature for 5 min. Subsequently, CD8 cells captured by the antibody were separated using a magnetic rack. + T cells, after discarding the uncaptured cell population, detach from the magnetic rack and collect CD8. + T cell populations were resuspended in RPMI 1640 complete medium containing 10 ng / mL porcine IL-2 and cultured statically in a cell culture incubator for 3 days before use.
[0040] 1×10 6 BMDCs stimulated by antigen pulses and 4 × 10 6 CD8 + T cell suspension was co-seeded into 6-well plates to establish a co-culture system, ensuring the cell culture environment contained 10 ng / mL porcine IL-2. On day 5, each co-culture well was aliquoted into two new wells of the 6-well plate, and an additional 2 mL of fresh culture medium (containing 10 ng / mL IL-2) was added to each well. Cells were cultured for another 3 days. On day 8, cells were collected by gentle aspiration, mixed thoroughly, and 20 μL of the mixture was mixed with an equal volume of trypan blue staining solution to label the cells. The number of viable cells was then counted using a cell counter. CD8 cells were isolated using the magnetic bead method described above. + T cells are antigen-specific T cells.
[0041] 3. ASFV RNA polymerase protein recombinant T cell epitope peptide stimulates specific CD8 + T cell proliferation Cells were distributed at 5 × 10⁶ cells per well in a 96-well plate. 4 Cells were seeded into plates and allowed to rest overnight. Then, peptides were added to the plates at final concentrations of 5 μg / mL, 10 μg / mL, and 20 μg / mL, respectively, with LPS as a positive control. After incubation in a cell culture incubator for 24 h and 48 h, 10 μL of CCK-8 solution was added to each well. After incubation in a cell culture incubator for 4 h, OD450 was measured. The changes in cell viability in each treatment group were calculated with the normal culture medium group as a control.
[0042] 4. Experimental Results Experimental results are as follows Figure 4 and 5As shown, the cell proliferation-inducing ability of P6 exhibited a time-dependent effect, and within the experimental concentration range, the 40 μg / mL concentration showed the most significant stimulation of T cell proliferation, with proliferation induction rates 1.36 and 1.62 times higher than the control group at 24 h and 48 h, respectively. The cell proliferation-inducing ability of P8 also showed a time- and concentration-dependent effect, with the 160 μg / mL concentration treatment group showing proliferation induction rates 1.14 and 1.54 times higher than the control group at 24 h and 48 h, respectively. Therefore, the peptides in this invention possess the ability to induce specific T cell proliferation.
[0043] In summary, this invention provides a linear tandem antigenic epitope polypeptide of African swine fever virus (ASFV) RAN polymerase and its application. The amino acid sequence of the linear tandem antigenic epitope polypeptide is shown in SEQ ID NO.4 or SEQ ID NO.5. The linear tandem antigenic epitope polypeptide is obtained by linearly tandemly connecting epitope polypeptides NP1249L-1, H359L-1, and H359L-2. Epitope polypeptides NP1249L-1, H359L-1, and H359L-2 are obtained through screening and have the functions of inducing ASFV-specific T cells, assisting in the control of ASFV infection, and clearing the virus. Experimental results show that the epitope polypeptide has the ability to induce ASFV-specific T cell responses, providing a theoretical basis for the subsequent development of polypeptide vaccines and diagnostic agents based on ASFV protein-derived epitopes.
[0044] The above description describes specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and these modifications or substitutions should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.
Claims
1. A linear tandem antigenic epitope polypeptide of African swine fever virus RAN polymerase, characterized in that, The amino acid sequence of the linear tandem antigenic epitope polypeptide is shown in SEQ ID NO.4 or SEQ ID NO.
5.
2. The linear tandem antigenic epitope polypeptide according to claim 1, characterized in that, The linear tandem antigenic epitope polypeptide is obtained by linearly tandemly connecting one or more epitope polypeptides NP1249L-1, H359L-1, and H359L-2.
3. The linear tandem antigenic epitope polypeptide according to claim 2, characterized in that, The amino acid sequence of the epitope polypeptide NP1249L-1 is shown in SEQ ID NO.1, the amino acid sequence of the epitope polypeptide H359L-1 is shown in SEQ ID NO.2, and the amino acid sequence of the epitope polypeptide H359L-2 is shown in SEQ ID NO.
3.
4. A nucleic acid molecule, characterized in that, The nucleic acid molecule described herein can encode the linear tandem antigenic epitope polypeptide of any one of claims 1-3.
5. A recombinant vector, characterized in that, It contains the nucleic acid molecule as described in claim 4.
6. An antigen-presenting cell, characterized in that, The antigen-presenting cells are sensitized with the linear tandem antigenic epitope polypeptide as described in claim 1.
7. An African swine fever virus antigen, characterized in that, The African swine fever virus antigen is prepared by coupling the linear tandem antigenic epitope polypeptide of any one of claims 1-3 with a carrier protein.
8. The application of the linear tandem antigenic epitope polypeptide according to any one of claims 1-3, characterized in that, Used for the preparation of peptide vaccines against African swine fever virus, preparation of specific antibodies against African swine fever virus, and preparation of reagents or kits for the diagnosis or detection of African swine fever virus.